The 5-HT system in the gut has proved most reliable for therapeutic manipulation of colonic motility. Clinical Researchers focus on the abundant mucosal 5-HT (>95% of body's 5-HT in enterocromaffin cells-EC) and tend to ignore neuronal 5-HT (~2%) whose function is unclear. 5-HT is synthesized by the rate limiting enzyme tryptophan hydroxylase (TPH), TPH1 in EC cells and TPH2 in neurons. Myenteric 5-HT (TPH2) interneurons are important in development of enteric neurons and ICC and guard against inflammatory damage. Myenteric 5-HT neurons innervate the entire ENS, glial cells and interstitial cells of Cajal, suggesting the novel hypothesis that they are a kind of Central Processing Unit (CPU) coupling motility and secretion. We hypothesize that myenteric 5-HT neurons are essential for driving several colonic motor behaviors including tonic inhibition of the muscle and colonic migrating motor complexes (CMMCs). We demonstrate that CMMCs in mice and primates are powerful rhythmic peristaltic contractions comparable in frequency and duration to high amplitude propagating contractions (HAPCs) in humans. Unlike tonic inhibition, CMMCs/HAPCs are normally abolished by removing the mucosa suggesting that both neuronal and mucosal 5-HT are synergistic and normally coupled together by mucosally projecting intrinsic primary afferent neurons (IPANs), which are activated by mucosal release of EC cell 5-HT. To generate CMMCs/HAPCs, tonic inhibition has to be switched off. We propose that activation of myenteric glial cells also activated by 5-HT neurons are necessary to switch between these motor activities. Glial cells release prostaglandins (PGE2) to suppress inhibitory motor neurons to allow for the CMMC/HAPC contraction by excitatory motor neurons. We will demonstrate that 5-HT neurons are essential in in regulating and switching between tonic inhibition and CMMCs/HAPCs. AIM 1. Determine the role of 5-HT (TPH2) neurons, IPANs and cholinergic motor neurons in CMMC/HAPC generation. AIM 2: Determine the role of 5-HT neurons and glia in tonic inhibition and disinhibition. AIM 3. To determine how rhythmic CMMC like contractions occur in partially obstructed mice (POM-ST) and TPH1 KO mice that lack mucosal 5-HT release. To address the critical role of 5-HT neurons we will use novel Optogenetic Technologies (?Breakthrough of the Decade?) in mice where neuronal or glia marker proteins are genetically coupled to either GCaMP3 (genetically encoded Ca2+ indicator responding to electrical activity) or to channel Opsins (cation or anion channels) that can be activated or inhibited by light rather than pharmacologically. We have generated WNT1-GCaMP (labels all enteric neurons and glia); ChAT-GCaMP3 (all cholinergic motor and interneurons), nNOS-GCaMP3 (inhibitory motor neurons/some interneurons) and TPH2-GCaMP3 (5-HT neurons). We have inserted Channelrhodopsin (ChR-2) and Halorhodpsin (HR) into 5-HT (TPH2) neurons that can be depolarized or hyperpolarized by blue and yellow light respectively, allowing their behavior to be easily manipulated during tonic inhibition and CMMCs. Our findings will be confirmed in segments of primate and human colon.